Conventional methods for transforming monocots include electroporation, the polyethylene glycol method (PEG method), the particle gun method and so on.
The electroporation method is a method in which protoplasts and the desired DNA are mixed, and holes are formed in the cell membranes by electric pulse(s) so as to introduce the DNA into the cells, thereby transforming the cells. This method currently has the highest reproducibility of the conventional methods and various genes have been introduced into monocots, especially into rice plants by this method (Toriyama K. et al., 1988; Bio/Technol. 6:1072–1074, Shimamoto K. et al., 1989; Nature 338:274–276, Rhodes C. A. et al., 1989; Science 240:204–207). However, this method has the problems that 1) it can be applied only to plant species for which a system for regenerating plants from protoplasts has been established, 2) since it takes several months to regenerate plants from the protoplasts, a long time is required to obtain transformants, and 3) since the culture period is long, the frequency of emergence of mutants during culture is accordingly high, so that the probability of obtaining normal transformants is decreased.
The PEG method is a method in which the desired gene and protoplasts are mixed and the mixture is treated with PEG, thereby introducing the gene into the protoplasts. This method is different from the electroporation method in that PEG is used instead of electric pulses. The efficiency of introducing the gene is thought to be somewhat lower than by the electroporation method. Although there is a report that transformants were obtained by this method, this method is not widely used. Since protoplasts are used, this method has the same problems as the electroporation method (Zhang W. et al., 1988; Theor. Appl. Genet. 76:835–840, Datta S. K. et al., 1990; Bio/Technol. 8:736–740).
The particle gun method is a method in which the desired gene is attached to fine metal particles and the metal particles are shot into cells or tissues at a high speed, thereby carrying out the transformation. Thus, according to this principle, transformation may be performed on any tissues. Therefore, this method is effective for transforming plant species for which a system for regenerating plants from protoplasts has not been established. The efficiency of transformation varies depending on the selection method after the gene was shot into the plant cells. There are no data which compare the efficiency of this method with that of the electroporation method (Gordon-Kamm W. J. et al., 1990; Plant Cell 2:603–618, Fromm M. E. et al., 1990; Bio/Technol. 8:833–839, Christou P. et al., 1991; Bio/Technol. 9:957–962).
Other methods for transforming plants include 1) culturing seeds or embryos with DNA (Topfer R. et al., 1989; Plant Cell 1:133–139, Ledoux L. et al., 1974 Nature 249:17–21); 2) treatment of pollen tubes (Luo and Wu 1988; Plant Mol. Biol. Rep. 6:165-), 3) a liposome method (Caboche M. 1990; Physiol. Plant. 79:173–176, Gad A. E. et al., 1990:177–183) and 4) a microinjection method (Neuhaus G. et al., 1987; Theor. Appl. Genet. 75:30–36). However, these methods have problems in the efficiency of transformation, reproducibility or applicability, so that these methods are not commonly used.
On the other hand, a method for introducing a gene using the Ti plasmid of bacteria belonging to the genus Agrobacterium as a vector is widely used for transforming dicotyledonous plants (“dicots”) such as tobacco, petunia, rape and the like. However, it is said that the hosts of the bacteria belonging to genus Agrobacterium are restricted to dicots and that monocots are not infected by Agrobacterium (De Cleene M. 1976; Bot. Rev. 42:389–466).
As for transformation of monocots by Agrobacterium, although transformation of asparagus (Bytebier B. et al., 1987: Proc. Natl. Acad. Sci. USA, 84:5345–5349) and of Dioscorea bulbifera (Schafew et al., 1987; Nature 327:529–532) has been reported, it is said that this method cannot be applied to other monocots, especially to plants belonging to family Gramineae (Potrykus I. 1990; Bio/Technol. 8:535–543).
Grimsley et al. (1987: Nature 325:177–179) reported that T-DNA of Agrobacterium in which DNA of maize streak virus was inserted was inoculated to the apical meristem of maize plants and infection of the plants by maize streak virus was confirmed. Since infection symptoms were not observed when the DNA of only the maize streak virus was inoculated, they interpreted the above-mentioned result as a piece of evidence showing that Agrobacterium can introduce DNA into maize. However, since it is possible that a virus replicates even if it is not incorporated into the nuclear genome of the plant cell, their result does not show that any T-DNA was incorporated into the nucleus. They subsequently reported that the infection efficiency is the highest when the virus is inoculated to the apical meristem in the shoot apex of the maize (Grimsley et al., 1988: Bio/Technol. 6:185–189), and that the virC gene in the plasmid of Agrobacterium is indispensable to the infection (Grimsley et al., Mol. Gen. Genet. 217:309–316).
Gould J. et al. (1991; Plant Physiol. 95:426–434) inoculated super-virulent Agrobacterium EHA1 having a kanamycin-resistance gene and a GUS gene to shoot apices of maize after injuring the shoot apices with a needle, and selected for kanamycin resistant shoot apices. As a result, plants having resistance to kanamycin were obtained. They confirmed by Southern blotting analysis that some of the seeds of the subsequent generation of the selected plants had the introduced gene (a so-called “chimera phenomenon”).
Mooney P. A. et al., (1991; Plant Cell, Tissue, Organ Culture 25:209–218) tried to introduce kanamycin-resistant gene into embryos of wheat using Agrobacterium. The embryos were treated with an enzyme to injure the cell walls, and then Agrobacterium was inoculated. Among the treated calli, although very small number of calli which were assumed to be transformants grew, plants could not be regenerated from these calli. The existence of the kanamycin-resistance gene was checked by Southern blotting analysis. In all of the resistant calli, change in the structure of the introduced gene was observed.
Raineri et al. (1990; Bio/Technol. 8:33–38) inoculated super-virulent Agrobacterium A281 (pTiBo542) to 8 varieties of rice after injuring the scutella of the rice plants. Growth of tumor-like tissues was observed in two varieties, Nipponbare and Fujisaka 5. Further, an Agrobacterium containing a plasmid having a T-DNA from which a hormone-synthesizing gene was removed and instead, a kanamycin-resistance gene and GUS gene were inserted therein was inoculated to embryos of rice. Growth of kanamycin-resistant calli was observed. Although expression of the GUS gene was observed in these resistant calli, transformed plants could not be obtained from the calli. Raineri et al. interpreted these results as showing that the T-DNA was introduced into rice cells.
Thus, although experimental results which suggest that introduction of genes into the plants belonging to family Gramineae such as rice, maize and wheat can be attained by using Agrobacterium have been reported, results fully convincing of reproducibility, introduction efficiency and confirmation of the introduction of the gene have not been obtained (Potrykus I. 1990; Bio/Technol. 8:535–543).
As mentioned above, introduction of genes into plants belonging to family Gramineae is now mainly carried out by the electroporation method. However, with this method, since protoplasts are used, a long time and much labor are required to obtain regenerated plants. Further, there is a danger that mutants may emerge at a high frequency due to the long culturing period. Still further, this method cannot be applied to plants such as maize for which a system for regenerating plants from protoplasts has not been established. In view of this, as mentioned above, as for maize, it has been tried to use the apical meristem. However, the operation for isolating the apical meristem requires much labor and it is not easy to prepare apical meristem in a large amount.